MAMMALIAN NEURAL PLATE BORDER STEM CELLS CAPABLE OF FORMING NEURAL TUBE AND NEURAL CREST CELL LINEAGES INCLUDING CENTRAL AND PERIPHERAL NEURONS

摘要

The present invention relates to a method for producing mammalian neural plate border stem cells (NPBSCs), comprising: (a) differentiation of mammalian pluripotent stem cells by (a-i) culturing mammalian pluripotent stem cells in pluripotent stem cell medium for about 24 to about 96 hours, wherein the pluripotent stem cell medium comprises: (i) an inhibitor of the activin/TGF-β signalling pathway; (ii) an inhibitor of the BMP signalling pathway; (iii) an activator of the canonical WNT signalling pathway; and (iv) an activator of the Hedgehog signalling pathway; subsequently (a-ii) culturing the cells obtained in step (a-i) for about 24 to about 96 hours in a neural medium, wherein the neural medium comprises: (i) an inhibitor of the Activin/TGF-β signalling pathway; (ii) an inhibitor of the BMP signalling pathway; (iii) an activator of the canonical WNT signalling pathway; and (iv) an activator of the Hedgehog signalling pathway; subsequently (a-iii) culturing the cells obtained in step (a-ii) for about 24 to about 96 hours in a neural medium, wherein the neural medium comprises: (i) an activator of the canonical WNT signalling pathway; (ii) an activator of the Hedgehog signalling pathway; and (iii) an inhibitor of oxidation; and (b) plating the obtained differentiated mammalian pluripotent stem cells in NPBSCs expansion medium, wherein the NPBSCs expansion medium comprises (i) an activator of the canonical WNT signalling pathway; (ii) an activator of the Hedgehog signalling pathway; and (iii) an inhibitor of oxidation; and expanding the cells in the NPBSCs expansion medium for about 24 to about 96 hours; (c) splitting the cells obtained in (b) and further expanding the cells in the NPBSCs expansion medium; and (d) repeating step (c) at least two times. The present invention further relates to neural plate border stem cells obtainable by the method of the invention and the use of the cells of the invention in medicine.

主权项

1. A method for producing mammalian neural plate border stem cells (NPBSCs), comprising:
(a) differentiation of mammalian pluripotent stem cells by
(a-i) culturing mammalian pluripotent stem cells in pluripotent stem cell medium for about 24 to about 96 hours, wherein the pluripotent stem cell medium comprises:
(i) an inhibitor of the activin/TGF-β signalling pathway;(ii) an inhibitor of the BMP signalling pathway;(iii) an activator of the canonical WNT signalling pathway; and(iv) an activator of the Hedgehog signalling pathway; subsequently(a-ii) culturing the cells obtained in step (a-i) for about 24 to about 96 hours in a neural medium, wherein the neural medium comprises:
(i) an inhibitor of the activin/TGF-β signalling pathway;(ii) an inhibitor of the BMP signalling pathway;(iii) an activator of the canonical WNT signalling pathway; and(iv) an activator of the Hedgehog signalling pathway; subsequently(a-iii) culturing the cells obtained in step (a-ii) for about 24 to about 96 hours in a neural medium, wherein the neural medium comprises:
(i) an activator of the canonical WNT signalling pathway;(ii) an activator of the Hedgehog signalling pathway; and(iii) an inhibitor of oxidation; (b) plating the obtained differentiated mammalian pluripotent stem cells in NPBSCs expansion medium, wherein the NPBSCs expansion medium comprises
(i) an activator of the canonical WNT signalling pathway;(ii) an activator of the Hedgehog signalling pathway; and(iii) an inhibitor of oxidationand expanding the cells in the NPBSCs expansion medium for about 24 to about 96 hours; (c) splitting the cells obtained in (b) and further expanding the cells in the NPBSCs expansion medium; and (d) repeating step (c) at least two times.